Ginkgo Bilboa flavonoid extract which is terpene-free and has a high flavonoid heteroside content

ABSTRACT

The invention concerns a  Ginkgo biloba  leaf flavonoid extract and more specifically an extract which is terpene free and/or has a high flavonoid heteroside content. The invention also concerns a flavoring compound containing such an extract and the use of this extract as a flavoring ingredient. The invention finally concerns a process for obtaining a substantially terpene-free  Ginkgo biloba  leaf flavonoid extract.

This application is a 371 of PCT/FR96/00653 filed Apr. 29, 1996.

The invention concerns a flavonoid extract of Ginkgo biloba and morespecifically an extract either substantially free of terpenes or with ahigh content of flavonoid heterosides, or free of terpenes and with ahigh content of flavonoid heterosides. This extract may advantageouslybe used as a flavoring agent. The invention likewise concerns aflavoring composition comprising such an extract and the use of thisextract as a flavoring ingredient.

Applications of extracts of Ginkgo biloba in the field of medicine andcosmetics are well known. The extract EGb-761 is perhaps the best knownin the medical field (The extract of Ginkgo biloba [EGb-761], La PressMédicale, 1986, Vol. 31, Special Number, Masson Publishing Co.). Thisextract primarily includes two families of substances: the flavonoid andterpene substances. New extracts were defined which, in an unexpectedway, modify the organoleptic [sensory] properties of certain foods suchas drinks, dairy products, [and] sweets.

One of the aspects of the present invention therefore has as its objectextracts which do not comprise any or only a small quantity of terpenes(ginkgolides and bilobalides) which have a high degree of therapeuticactivity. Moreover, it has been found to be of interest to obtainextracts enriched with flavonoid substances: these are essentially themono-, di-, and tri-glucosides of Kaempferol, of Quercetine, and ofIsorhamnetine with glucose and with rhamnose.

The invention also has as its object a flavonoid extract of Ginkgobiloba leaves free of terpenes. This means that the extract comprisesflavonoid heterosides and small quantities of terpenes or no terpenes.If the extract comprises terpenes, the terpene content is a maximum of1%, preferably a maximum of 0.5%.

The invention likewise has as its object a flavonoid extract with a highflavonoid heteroside content. This means that the extract comprises from28 to 35% flavonoid heterosides, and preferably 28 to 32%. Such extractsare obtained preferably from the cut leaves of young Ginkgo bilobatrees.

The invention likewise has as its object a flavonoid extract free ofterpenes and with a high content of flavonoid heterosides. This meansthat the extract is free of terpenes and comprises from 28 to 35%flavonoid heterosides, preferably from 28 to 32%.

More especially, the invention has as its object a flavonoid extractcomprising maximally 1% terpenes and from 28 to 25% flavonoidheterosides. Preferably the extract comprises maximally 0.5% terpenesand from 28 to 32% flavonoid heterosides.

A flavonoid extract with a high content of flavonoid heterosides may beobtained by extraction under partial vacuum with an acetone-watermixture. After stages of delipidation, elimination of undesirablesubstances with various solvents and by precipitation, the extractsolution is concentrated and the extract is dried in vacuo.

The invention likewise has as its object a procedure for preparing anextract free of terpenes as defined above. This procedure includesseveral extraction stages of extract of Ginkgo biloba leaves withsolvents, characterized in that one of the extraction stages is adeterpenation stage and the solvent used is a compound of formulaRC(O)OR′ in which R and R′ represent independently a lower alkyl aloneor mixed with a saturated aliphatic hydrocarbon comprises at least 5carbon atoms. The extraction stage may be effected at any stage of theprocess. The solvent used during the determination stage comprisespreferably from 0 to 20% saturated aliphatic compound.

Extraction stages other than the deterpenation stage are known in theliterature, especially in Patents EP 431535, EP 431536, EP 360556, andEP 324197. These patents are incorporated by reference into the presentapplication.

In the above-indicated definitions, the term lower alkyl representspreferably a linear or branched alkyl radical having from 1 to 6 carbonatoms and in particular an alkyl radical having from 1 to 4 carbon atomssuch as the methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, and tert-butyl radicals. Solvents of formula RC(O)OR′ inwhich the R and R′ radicals represent methyl, ethyl, or propyl, andespecially ethyl acetate, are preferably used.

The saturated aliphatic hydrocarbon may be chosen from among hexane,heptane, [or] octane. Heptane is preferably used.

The invention likewise has as its object a procedure for preparing anextract enriched with flavonoid heterosides as defined above. Thisprocedure includes several stages of extraction from the leaves ofGinkgo biloba with solvents, characterized in that one of the extractionstages is a stage of enrichment with flavonoid heterosides and that thesolvent used, in a minimum quantity, is an alcohol, alone or mixed witha ketone, preferably acetone. The extraction stage may be carried out atany stage of the procedure. The alcohol used is preferably a loweralcohol such as methanol, ethanol, propanol, [or] butanol, andpreferably butanol. The amount of solvent used may be from 3 to 12parts, and preferably in the lower portion of this range.

An example is described below in the experimental part; the order of theextraction stages with the ethyl acetate/heptane mixture and theacetone-butanol mixture may be reversed.

An extract according to the invention simultaneously modifies theolfactory profile and the taste profile of the product tested. By way ofits organoleptic properties, this extract may be used for thepreparation of aromatic compositions of various types. It may be usedalone or in association with other flavoring ingredients currently used.Thus, the use of such a flavonoid extract is anticipated in foodproducts. As food products, one may cite dairy products such as forexample yogurts, refreshing or nutritious drinks, and more especiallyrefreshing drinks without alcohol such as orange drinks or tonics basedon quinine, but likewise sweetened products such as for example chewinggum.

The amount used in a flavoring composition or a flavored product mayvary over a wide range of values: it is understood that this valuedepends on the product into which the flavor or the flavoringcomposition is incorporated, but also on the nature and amount of otherconstituents of the flavoring composition and upon the desired effect.

The flavonoid extract according to the invention is used inconcentrations ranging from 0.001 to 0.1% by weight relative to thetotal weight of the flavored product, preferably between 0.001 and0.05%. In the case of dairy products, the flavonoid extractconcentration may range from 0.005 to 0.045%. In the case of productssuch as chewing gums, the flavonoid extract concentration may range from0.01 to 0.04%. In the case of food products such as refreshing ornutritious drinks, the flavonoid extract concentration may range from0.002 to 0.02%.

The following examples are presented to illustrate the invention and arenot in any case to be considered as limiting the scope of the invention.

EXAMPLE 1 Procedure for Obtaining a Flavonoid Extract

The leaves of Ginkgo biloba are extracted with 6 to 12 parts (preferably8) of water comprising 60% acetone at 50-60 degrees C. and the solutionis concentrated so as to reduce the percentage of acetone to less than3%. This solution is cooled and the lipids are eliminated bydecantation. The aqueous solution is extracted with 2 to 5 parts ofethyl acetate comprising 0 to 20% heptane. The resulting solution isextracted with a minimum amount of an acetone-butanol mixture (0 to 15%acetone) in the presence of ammonium sulfate. The organic phase isconcentrated; after adding ethanol, the solution is again concentrated.After a new dilution with ethanol, the solution is cooled and theinsoluble precipitates are filtered out. The resulting solution isconcentrated, dried, and finally pulverized to recover the flavonoidextract in the form of a homogeneous powder.

EXAMPLE 2 Sensory Analysis

A sensory analysis of a flavonoid extract as obtained in Example 1 wascarried out for a certain number of food products (dairy products,refreshing drinks, and chewing gums) with or without flavonoid extract.

Sixty tasters participated in this analysis. For each sensory item(acidity, bitterness, sweetness, saltiness) each taster indicated anintensity grade from 0 to 5 corresponding to an increasing degree ofevaluation of the product tested.

EXAMPLE 2a Dairy Products

Q Conventional solid yogurt Light solid & yogurt (mg/pot) 0 10 25 0 1025 Acidity 4 3 2.5 3 2.5 3 Bitterness 2.5 2.5 2.5 2 2.5 1.5 Sweetness 12.5 2 2.5 2 2.5 Saltiness 1 1.5 2 3 2 2 Q = amount of extract

Q Conventional solid yogurt Light solid & yogurt (mg/pot) 0 10 25 0 1025 Acidity 4 3 2.5 2 2.5 2 Bitterness 2 2 2.5 2 2.5 2 Sweetness 2 2.5 24 3 2 Saltiness 2 2.5 2 2 2.5 2

Q Sweetened natural drinking yogurt (mg/l) 0 50 100 Acidity 3 3.5 3Bitterness 1 1.5 1.5 Sweetness 3.5 3 3 Saltiness 1 1 1

The natural astringency of the extract balances well the fundamentalflavors of dairy products by attenuating the acidity of traditionalproducts. The extract appeared to be very complementary with the dairyand fruit characters.

EXAMPLE 2b Refreshing Drinks

Q Quinine-based tonic drink Orange drink (mg/l) 0 50 100 0 50 100Acidity 2 2.5 3 2.5 3 3.5 Bitterness 3 2.5 3 2 3.5 4 Sweetness 2.5 2.53.5 3.5 2.5 4 Saltiness 0.5 0.5 0.5 2 1 1

The astringency of the extract is harmoniously blended with that of thedrinks, and more especially with that of quinine grade drinks and/orbitter barks such as Indian Tonic®.

EXAMPLE 2 Chewing Gums

Mentholated Chlorophyll Other mentholated chewing gum chewing gumchewing gum U (mg/unit) 0 3 6 0 6 12 0 3 6 Freshness 4 2 1 2 3 1 2 3 1Tartness 4 3 1 2 2 1 2 2 1 Sweetness 3 3.5 4 2 3 4 3 4 4 Herbal flavor 02 4 2 3 4 1 2 4 U = unit

EXAMPLE 3 Descriptive Test

This test was conducted with several varieties of yogurts. Sixtytasters, consumers of natural yogurts, participated in the test. Themethod used was a comparative test of sensory profiles. Thequestionnaire contained 9 to 10 descriptive items according to the“solid” or “mixed” variety tested. Each taster indicated on a 10 cmnonstructured scale for each item the perceived intensity and this wascarried out successively for all of the products. The results obtainedare the following:

EXAMPLE 3a Product Tested: Solid Yogurts 0% of Fatty Matter, with orwithout the Addition of 0.04% of a Flavonoid Extract as Obtained inExample 1.

Variety with extract Variety without extract Standard StandardDescriptor Average deviation Average deviation t S Firmness 6.68 1.356.58 1.52 0.81 Overall inten- 5.00 1.72 4.77 1.72 1.42 sity of aromaSmooth consis- 6.54 2.03 6.34 1.79 1.18 tency in the mouth Denseness4.90 1.54 4.73 1.73 0.97 Fatty sensation 2.31 1.98 2.04 1.91 1.95 in themouth Fruity taste 2.65 2.10 0.70 1.02 8.36 1% Fermented taste 2.99 2.333.22 2.13 1.28 Acid taste 2.74 2.07 2.67 1.98 0.33 Bitter taste 2.071.82 1.42 1.31 3.95 1% t = Student's “T” on paired series; S = degree ofsignificance

The variety with the flavonoid extract is distinguished in a highlysignificant way as having a more pronounced fruity taste and a moreintensely bitter taste than the variety without flavonoid extract.

EXAMPLE 3b Product Tested: Fatty Solid Yogurts, with or without theAddition of 0.04% Flavonoid Extract as Obtained in Example 1.

Variety with extract Variety without extract Standard StandardDescriptor Average deviation Average deviation t S Firmness 7.65 1.607.38 1.36 2.06 5% Overall inten- 4.85 1.85 4.81 1.92 0.27 sity of aromaSmooth consis- 7.01 1.99 6.87 2.12 0.92 tency in mouth Denseness 6.262.05 5.46 1.91 4.92 1% Fatty sensation 4.17 2.08 3.54 1.95 3.59 1% inmouth Fruity taste 2.25 1.99 0.84 1.19 6.04 1% Fermented taste 2.62 2.062.66 2.19 0.24 Add taste 2.62 1.72 2.61 1.59 0.05 Bitter taste 1.69 1.551.53 1.49 0.88 t = Student's “T” in paired series; S = Significance

The variety with the flavonoid extract is distinguished in a highlysignificant way as having a denser consistency in the mouth, a moreintense sensation of fat in the mouth, and a more pronounced fruitytaste and, in a significant way, as having a firmer consistency on thespoon than the variety without flavonoid extract.

EXAMPLE 3c Product Tested: Fat Mixed Yogurts with or without theAddition of 0.04% Flavonoid Extract as Obtained in Example 1.

Variety with extract Variety without extract Standard StandardDescriptor Average deviation Average deviation t S Denseness 5.29 1.715.22 1.71 0.56 on spoon Ropy consis- 3.52 1.91 3.50 1.80 0.20 tency onthe spoon Overall inten- 4.36 1.69 4.32 1.68 0.27 sity of aroma Smoothsensa- 8.23 1.48 8.17 1.63 0.66 tion in mouth Denseness 3.99 1.96 4.062.04 0.45 Fatty sensa- 3.72 2.01 3.42 1.82 1.77 tion in mouth Fruitytaste 2.60 1.92 1.09 1.31 5.97 1% Fermented taste 3.19 2.20 2.94 2.271.11 Acid taste 3.10 2.00 2.47 1.71 2.63 5% Bitter taste 2.00 1.78 1.531.57 2.35 5% t = Student's “T” in paired series; S = Significance

The variety with the flavonoid extract is distinguished in a highlysignificant way as having a more pronounced fruity taste and, in asignificant way, as having a more acidic taste and a more bitter tastethan the variety without flavonoid extract.

What is claimed is:
 1. A flavonoid extract from the leaves of the Ginkgobiloba, containing at most 1% of terpenes and 28 to 35% by weight offlavonoid heterosides.
 2. An extract as defined in claim 1, comprisingless than 0.5% terpenes.
 3. An extract as defined in claim 1, comprisingfrom 28 to 32% flavonoid heterosides.
 4. A process for preparing anextract as defined in claim 1, comprising extracting several times theleaves of the Ginkgo biloba with solvents, wherein one of the extractionstages is a deterpenation stage using a solvent of formula RC(O)OR′ inwhich R and R′ are individually lower alkyl, alone or mixed with asaturated aliphatic hydrocarbon of 1 to 5 carbon atoms.
 5. The processof claim 4, wherein the solvent used for the deterpenation stage isselected from the group consisting of methyl, ethyl, and propyl acetate,and comprises 0 to 20% saturated aliphatic hydrocarbon.
 6. A flavoringcomposition comprising as flavoring ingredient an extract according toclaim 1 alone or in association with other flavors.
 7. A productflavored with a composition according to claim
 6. 8. A product accordingto claim 7 which is a dairy product.
 9. A product according to claim 7which is a drink.
 10. A product according to claim 7 which is asweetened product.
 11. A food product containing the flavonoid extractof claim 1 as a food flavoring.
 12. A yogurt containing the flavonoidextract of claim 1 to provide a pronounced fruity flavor and a firmerconsistency.
 13. A nutritious non-alcoholic drink containing theflavonoid extract of claim 1 as a flavoring agent.
 14. A product ofclaim 8 where the dairy product is yogurt.
 15. A product of claim 10which is chewing gum.